Much research has been devoted in recent years to the conjugation of therapeutic agents as payloads to peptides and proteins for a wide range of applications. The protein or peptide itself may have therapeutic properties, and/or it may be a binding protein. In addition, conjugation to polymers has been used to improve the properties of certain therapeutic agents. For example, water soluble, synthetic polymers, particularly polyalkylene glycols, are widely used to conjugate therapeutically active peptides or proteins. These therapeutic conjugates have been shown to alter pharmacokinetics favourably by prolonging circulation time and decreasing clearance rates, decreasing systemic toxicity, and in several cases, displaying increased clinical efficacy. The process of covalently conjugating polyethylene glycol, PEG, to proteins and other payloads is commonly known as “PEGylation”.
Binding proteins (i.e. proteins or peptides capable of binding to a binding partner on a target), particularly antibodies or antibody fragments, are frequently used in conjugates. For example, they may be conjugated to cytotoxic agents and chemotherapy drugs to produce antibody-drug conjugates (ADCs), allowing targeted delivery of such agents to specific tissues or structures, for example particular cell types or growth factors, minimising the impact on normal, healthy tissue and significantly reducing the side effects associated with chemotherapy treatments. Such conjugates have extensive potential therapeutic applications in several disease areas, particularly in cancer.
Kupchan et al., J. Am. Chem. Soc., 94, 1354 (1972) first isolated Maytansine from the bark of the African shrub Maytenus ovatus, where it was noted for its anti-leukemic properties. Maytansinoids, such as maytansinol and C-3 esters of maytansinol were found to be made by microbes (U.S. Pat. No. 4,151,042), mosses (Sakai et al, J. Nat. Prod., 51(5), 845-850, 1988), and could also be generated by synthetic routes (Kupchan et al., J. Med. Chem., 21, 31-37, 1978, Higashide et al., Nature 270, 721-722, 1977). Maytansine and maytansinol have the structures:

U.S. Pat. No. 4,190,580 described a method of synthesis of maytansinoids, while U.S. Pat. No. 4,260,608 identified a number of maytansine derivative compounds with potent anti-microbial and anti tumour activities as a result of their anti-mitotic effects upon cells. U.S. Pat. No. 4,260,608 disclosed maytansine derivatives with an optionally substituted alkyl group at the R3 position. Since then numerous structure-activity relationship studies have been performed to determine other potent structures of maytansinoids. Kawai et al., Chem. Pharm. Bull. 32(9), 3441-3451 (1984) published a number of structures that were modified at the C3 position of Maytansinol, which displayed potent anti-tumour effects in vivo. EP 0 004 466, U.S. Pat. No. 4,137,230 and WO 2012/061590 all describe novel maytansinoids. Various modifications of maytansinoids are described in US 2009/258870, Taft et al, Chem. Eur. J. 2012, 18, 880-886, and Harmrolfs et al, Beilstein J. Org. Chem. 2014, 10, 535-543.
Well-known maytansinoids include those known as DM1 and DM4 as described within U.S. Pat. No. 5,208,020 and WO2004/103272. These have the structures:

Ansamitocins are a sub-group of maytansinoids, the synthesis of which is described by Taft et al, ChemBioChem 2008, 9, 1057-1060. A well-known ansamitocin is AP-3, which has the structure:

Maytansinoids and maytansinoid esters were subsequently used as a cytotoxic payload within an ADC context within EP 0 425 235 and have been used extensively since, see for example Liu et al, Proc. Natl. Acad. Sci. USA, 93, 8618-8623 (1996); and Kieda et al, Clin. Cancer Res., 15(12), 2009. Widdison et al, J. Med. Chem. 49, 4392-4408 (2006) described the thiol-linked DM1 and DM4 maytansinoid payloads. These payloads were conjugated via a two-step process in which first a heterobifunctional, thiol-containing linker was reacted with lysine residues within the antibody, followed by conjugation of the thiol-containing payloads to the linker by disulfide exchange. An alternative means of producing ADCs by this two-step process is by use of a succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC) heterobifunctional linker, which has a succinimide group at one end to react with lysine residues within the antibody and a maleimide group at the other end to react with the thiol group of the maytansinoid. The commercially-available pharmaceutical Kadcyla® is an example of an ADC produced using an SMCC linker with a DM1 maytansinoid payload. Kadcyla® is currently one of the leading ADCs in the clinic and is used for the treatment of HER2-positive breast cancer.
WO 2014/064424 describes ADCs based on maytansines using a different specific technology to bind the drug to the antibody. Exemplified are conjugates derived from the reagent AHX-DM1 available commercially from Levena (Concortis), and having the structure:

There is still a need for novel, potent cytotoxic molecules that may be used within an ADC context. These payloads need to be conjugated efficiently with an antibody using a conjugation technology which allows more stable ADCs to be generated. There is also a need for ADCs which show greater efficacy or potency in relevant cell or animal models of cancers, or alternatively, show a similar level of efficacy/potency but a reduced amount of non-specific toxicity within the cell or test subject.
We have now found that certain novel maytansinoid compounds possess improved cytotoxic activity, and are particularly suited for inclusion in conjugates with binding proteins. The maytansinoids further have improved stability compared with comparator compounds. We have also found a novel method of synthesis which enables efficient preparation of these novel compounds as well as a novel and improved method of synthesis of other maytansinoids.